Downloaded from www.microbiologyresearch.org by IP: 54.157.13.203 On: Sun, 28 Feb 2016 07:37:10 Biofilm differentiation and dispersal in mucoid Pseudomonas aeruginosa isolates from patients with cystic fibrosis Sylvia M. Kirov, 1 Jeremy S. Webb, 2 3 Che Y. O’May, 1 David W. Reid, 1 Jerry K. K. Woo, 2 Scott A. Rice 2 and Staffan Kjelleberg 2 Correspondence Sylvia M. Kirov S.M.Kirov@utas.edu.au 1 School of Medicine, University of Tasmania Clinical School, 43 Collins St, Hobart, Tasmania 7001, Australia 2 School of Biotechnology and Biomolecular Sciences and Centre for Marine Biofouling and Bio-innovation, University of New South Wales, Sydney, NSW 2052, Australia Received 16 April 2007 Revised 26 June 2007 Accepted 4 July 2007 Intractable biofilm infections with Pseudomonas aeruginosa are the major cause of premature death associated with cystic fibrosis (CF). Few studies have explored the biofilm developmental cycle of P. aeruginosa isolates from chronically infected individuals. This study shows that such clinical isolates exhibit biofilm differentiation and dispersal processes similar to those of the better-studied laboratory P. aeruginosa strain PAO1 in the glass flow-cell (continuous-culture) biofilm model, albeit they are initially less adherent and their microcolonies are slower to develop and show heterogeneous, strain-specific variations in architecture. Confocal scanning laser microscopy combined with LIVE/DEAD viability staining revealed that in all CF biofilms bacterial cell death occurred in maturing biofilms, extending from the substratum to the central regions of mature microcolonies to varying degrees, depending on the strain. Bacteriophage activity was detected in the maturing biofilms of all CF strains examined and the amount of phage produced paralleled the degree of cell death seen in the biofilm. Some CF strains exhibited ‘seeding dispersal’ associated with the above phenomena, producing ‘hollowing’ as motile cells evacuated from the microcolony interiors as has been described for strain PAO1. Moreover, morphotypic cell variants were seen in the biofilm effluents of all CF strains. For those CF strains where marked cell death and seeding dispersal occurred in the microcolonies, variants were more diverse (up to five morphotypes) compared to those of strain PAO1 (two morphotypes). Given that variants of strain PAO1 have enhanced colonization traits, it seems likely that the similar biofilm dispersal events described here for CF strains contribute to the variability seen in clinical isolates and the overall persistence of the P. aeruginosa in the CF airway. INTRODUCTION Cystic fibrosis (CF), the most common inherited lethal genetic disease in Caucasian populations, results from mutations in the CF transmembrane conductance regu- lator (CFTR) gene. These mutations result in defective cyclic-AMP-regulated chloride channel activity that impairs pancreatic, pulmonary and intestinal functions (Wilschanski et al., 1995). In the lung, mucus secretions become depleted of paraciliary fluid in the lower respiratory tract, impairing the clearance of inhaled microbes, resulting in chronic infections and an exagger- ated inflammatory response that leads to progressive deterioration in lung function and ultimately premature death (Lyczak et al., 2002; Saiman & Siegel, 2004). The opportunistic bacterial pathogen Pseudomonas aeruginosa is the most important agent involved in chronic lung infection in CF patients (Burns et al., 2001; Lyczak et al., 2002). The CF lung environment favours conversion of this organism from a motile, planktonic form to a mucoid, sessile biofilm mode of growth (Costerton et al., 1999; O’May et al., 2006; Worlitzsch et al., 2002). Recognized stages in biofilm development elucidated in experimental model systems include initial attachment, the formation of multicellular structures (‘microcolonies’), and the matura- tion of microcolonies into thick, three-dimensional structures encased in an exopolymeric matrix and inter- spersed with water channels (Stoodley et al., 2002). In the Abbreviations: AHLs, N-acylhomoserine lactones; CF, cystic fibrosis; HAQs, 4-hydroxy-2-alkylquinolines; HHQ, 4-hydroxy-2-heptylquiniline; HSL, homoserine lactone; LCV, large-colony variant; PQS, Pseudomonas quinolone signal; QS, quorum sensing; SCV, small-colony variant. 3Present address: School of Biological Sciences, University of Southampton, Southampton SO17 1BJ, UK. Microbiology (2007), 153, 3264–3274 DOI 10.1099/mic.0.2007/009092-0 3264 2007/009092 G 2007 SGM Printed in Great Britain